Head and neck cancer (HNC) is the sixth most common malignancy worldwide, diagnosed twice as frequently in veterans. Radiation therapy (RT) is an important component of cancer treatment; however, its efficacy is limited by radioresistance, with the cancer sometimes returning within the treated area. Resistance to RT is, at least in part, mediated by adaptive signaling events induced by treatment. However, we do not yet understand the pathways used by cells to evade the cellular damage caused by RT. The long-term goal is to better understand, and subsequently target, the mechanisms of resistance that cancer cells use to evade a radiation-induced death. Combinatorial adaptive response therapy (CART) represents a novel platform that allows for the rapid and systematic identification of treatment combinations that overcome therapeutic resistance and result in synthetic lethality. CART takes advantage of Reverse Phase Protein Microarray Analysis (RPPA), providing a high throughput, sensitive, and quantitative approach to analyze differential protein expression to identify targets for combinatorial therapy. The applicability of the CART approach to RT has not previously been investigated. The central objective of this career development application is to develop myself as an independent translational scientist with expertise in HNC, and to become a leader in the field of translational oncology, with implementation of a CART approach to increase the efficacy of RT in 3D culture and xenograft models. The rationale for the proposed research is rationally combining newly identified systemic treatments, such as the glutaminase inhibitor, CB-839, with RT will result in maximal efficacy while minimizing potential toxicities. Guided by strong preliminary data implicating glutaminase as playing a role in adaptive resistance to RT, we will pursue three specific aims: First (SA1), we will determine whether the combination of RT with a glutaminase inhibitor (CB-839) results in decreased aerobic respiration and increased cell death in 2D and 3D culture. To pursue this, upregulated aerobic respiration pathways, including those catalyzed by glutaminase, will be selectively targeted alone or in combination with RT in 3D with analysis of proliferation and apoptosis markers. Seahorse technology will be used to assess aerobic respiration. Second (SA2), we will validate the efficacy of glutaminase inhibition by testing it both alone and in combination with RT in preclinical heterotopic cell line and patient- derived xenograft animal models. Finally (SA3), we will identify other novel HNC signaling pathways that are significantly altered by RT using RPPA. For this aim, spheroids grown from oral cavity tumor derived cell lines will be grown in 3D culture and subjected to non-lethal RT doses with protein levels assessed by RPPA to identify candidate target proteins that are differentially expressed with RT. This innovative approach uses a cutting-edge, high-throughput, sensitive, and quantitative method (RPPA) to identify entirely novel therapeutic targets to be used in combination with RT, at the protein level. The proposed research is significant because it is testing a rationally selected treatment (CB-839) to increase the efficacy of RT. These experiments will lay the groundwork for future clinical trials and are expected to identify additional unknown, yet effective treatment combinations. In sum, this proposal outlines a sophisticated, rational, and rapid approach to identifying and testing novel therapeutic targets which would be of disproportionate benefit to veterans battling head and neck cancer.